The presence of certain species of Bifidobacterium is commonly observed in breast-fed infants (Roger & McCartney, Microbiology 156: 3317-3328 (2010)), and a bifidobacterial-dominant micobiota is thought to be associated with beneficial health effects (Le Huerou-Luron et al., Nutr Res Rev 23: 23-36 (2010); Conroy et al., Curr Opin Allergy Clin Immunol 9: 197-201 (2009)). This enrichment has been in part explained by the ability of bifidobacteria to degrade and utilize human milk oligosaccharides (HMO) as a carbon source (Ward et al., Mol Nutr Food Res 51: 1398-1405 (2007)). HMOs are complex free structures that escape digestion by intestinal enzymes (Kunz et al., Annu Rev Nutr 20: 699-722 (2000)). Among infant-associated bifidobacteria, B. longum subsp. infantis (B. infantis) ATCC 15697 has been studied for its ability to consume HMO in vitro and in vivo (LoCascio et al., J Agric Food Chem 55: 8914-8919 (2007); Marcobal et al., Cell Host Microbe 10: 507-514 (2011); Sela et al., Proc Natl Acad Sci USA 105: 18964-18969 (2008); Sela et al., J Biol Chem 286: 11909-11918 (2011); Garrido et al., PLoS One 6: e17315 (2011); Sela et al., Applied and Environmental Microbiology (2011)).
A great variability in protein types and abundances is found in the breast milk of different mothers at different stages of lactation (Mitoulas et al., Br J Nutr 88: 29-37 (2002)). Milk proteins are readily utilized by the infant (Prentice et al., Acta Paediatr Scand 76: 592-598 (1987)), and can play critical functions in protection of the newborn. For example, human lactoferrin (hLF) is one of the most abundant proteins in human milk, and hLF or its derived peptides display broad antimicrobial and anti-inflammatory effects, among several biological activities (Gonzalez-Chavez et al., Int Antimicrob Agents 33: 301 e301-308(2009)).
Many human milk proteins, as well as virtually all secreted proteins in eukaryotes, are glycosylated (Froehlich et al., J Agric Food Chem 58: 6440-6448 (2010)). While milk caseins are O-linked glycosylated, lactoferrin and immunoglobulins contain N-linked glycans (Picariello et al., Proteomics 8: 3833-3847 (2008)). Asparagine-linked glycosylation is the most common post-translational modification of eukaryotic proteins (Apweiler et al., Biochim Biophys Acta 1473: 4-8 (1999)). N-linked glycosylation (N-glycosylation) plays a role in folding, secretion, and resistance to proteolysis (Weber et al., J Biol Chem 279: 34589-34594 (2004); Roth et al., Mol Cells 30: 497-506 (2010)), protein function, such as bacterial recognition (Mathias & Corthesy, J Biol Chem 286: 17239-17247 (2011)), intracellular signaling (Sun et al., J Biol Chem 281: 11144-11151 (2006)) and antigen binding and presentation (Ryan et al., J Exp Med 208: 1041-1053 (2011)).
Certain microorganisms, mostly pathogens, have also acquired the ability to release N-glycans from glycoproteins, e.g., for use as a carbon source (Renzi et al., PLoS Pathog 7:e1002118 (2011)) or to alter the biological function of certain glycoproteins such as immunoglobulins (Collin et al., Proc Natl Acad Sci USA 105: 4265-4270 (2008)). Bacterial Endo-β-N-acetylglucosaminidases (EC 3.2.1.96; endoglycosidases) are enzymes that cleave the N—N′-diacetyl chitobiose of the core pentasaccharide Man3GlcNAc2 found in all N-glycans (Varki, Essentials of glycobiology (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) 2nd Ed pp xxix, 784 p. (2009)). These enzymes belong to glycosyl hydrolase families GH18 or GH85. Prominent examples are EndoH from Streptomyces plicatus (Trimble & Maley, Biochem Biophys Res Commun 78: 935-944 (1977)), EndoE from Enterococcus faecalis (Collin & Fischetti, J Biol Chem 279: 22558-22570 (2004)) and EndoS from Streptococcus pyogenes (Allhorn et al., PLoS One 3:e1413 (2008)), while EndoD from Streptococcus pneumoniae (Muramatsu et al., J Biochem 129: 923-928 (2001)) is a member of GH85. Previously characterized GH18 and GH85 endoglycosidases are of limited substrate specificity, to either high mannose or complex N-glycans and some require additional exoglycosidases for complete cleavage of the N-glycan.
Provided herein are deglycosylating enzymes (endoglycosidases) that cleave N-glycans from glycoproteins, but with a broad substrate range, able to cleave high mannose, hybrid and complex N-glycans from N-glycoproteins. The deglycosylating enzymes are active on N-glycans with terminal fucosylation and/or sialylation, and/or core fucosylation, and in a broad range of conditions.